Identification of salt accumulating organisms from winery wastewater FINAL REPORT to GRAPE AND WINE RESEARCH & DEVELOPMENT CORPORATION Project Number: UA08/01 Principal Investigator: Paul Grbin Research Organisation: University of Adelaide Date: 22/09/10 1 Identification of salt accumulating organisms from winery wastewater Dr Paul R Grbin Dr Kathryn L Eales Dr Frank Schmid Assoc. Prof. Vladimir Jiranek The University of Adelaide School of Agriculture, Food and Wine PMB 1, Glen Osmond, SA 5064 AUSTRALIA Date: 15 January 2010 Publisher: University of Adelaide Disclaimer: The advice presented in this document is intended as a source of information only. The University of Adelaide (UA) accept no responsibility for the results of any actions taken on the basis of the information contained within this publication, nor for the accuracy, currency or completeness of any material reported and therefore disclaim all liability for any error, loss or other consequence which may arise from relying on information in this publication. 2 Table of contents Abstract 3 Executive Summary 4 Background 5 Project Aims and Performance Targets 6 Methods 7 Results and Discussion 11 Outcomes and Conclusions 23 Recommendations 24 Appendix 1: Communication Appendix 2: Intellectual Property Appendix 3: References Appendix 4: Staff Appendix 5: Acknowledgements Appendix 6: Budget Reconciliation 3 Abbreviations: COD: Chemical oxygen demand Ec: Electrical conductivity FACS: Fluorescence activated cell sorting HEPES: 4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid OD: Optical density PBFI: Potassium benzofuran isophthalate PI: Propidium iodide SAR: Sodium adsorption ratio WWW: Winery wastewater Abstract: In an attempt to find microorganisms that would remove salts from biological winery wastewater (WWW) treatment plants, 8 halophiles were purchased from culture collections, with a further 40 isolated from WWW plants located in the Barossa Valley and McLaren Vale regions. These organisms were assessed for their ability to accumulate intracellular salt (in particular potassium), remove colour, reduce chemical oxygen demand, change pH and lower the electrical conductivity of a synthetic wastewater. Furthermore, fluorescence activated cell sorting (FACS) was employed to separate those cells that could accumulate higher concentrations of intracellular potassium. The outcomes of this study indicate that the many halophiles naturally occurring in WWW exhibit a large phylogenetic diversity, and some accumulated large concentrations of intracellular potassium, therefore further investigation is warranted to determine the applicability of these organisms in WWW treatment. 4 Executive summary: Halophilic synthetic winery wastewater (WWW) was designed to isolate halophiles from WWW. A total of 40 isolates were grown under aerobic and anaerobic conditions. These isolates were identified using 16S rRNA gene sequence analysis, and a large phylogenetic diversity was observed. The ability of these isolates to accumulate intracellular salts (particularly potassium, but also sodium, magnesium and calcium) was determined and some isolates accumulated 5 – 20 times more than others. Isolates were also evaluated for their effects on pH and Ec, and reduction in colour and chemical oxygen demand (COD). Generally, isolates did not affect pH or Ec, however there were slight changes in colour (hue) and COD (decrease). To eliminate the bias imposed by culture dependant techniques, environmental samples were directly evaluated for intracellular potassium concentration using the fluorescence indicator PBFI with fluorescence activated cell sorting (FACS). Those that gave very strong PBFI signals were separated from the remainder of the population. The DNA was then extracted, the 16S rRNA gene PCR amplified, amplicons separated by insertion into a plasmid and then identified by gene sequencing. Five WWW samples were examined by FACS/clone library. Four of the five clone libraries were dominated by a single species, Burkholderia phytofirmans strain PsJN. This study suggests that halophiles naturally occur in WWWs, with some (Halomonas, Psychrobacter and Arthrobacter sp.) exhibiting the potential to accumulate large concentrations of intracellular potassium. 5 Background: High salt content often persists throughout the biological treatment of WWW and can remain too high to allow its use as recycled irrigation water for grapevines. In 2008, 520,393 ML of water was used for the irrigation of grapevines across Australia, with only 0.9% of that being recycled water (Australian Grape and Wine Industry 2008). The build up of salts, especially sodium and potassium, limits the application of WWW to land as it leads to poor soil health affecting plant growth and vitality. Although potassium is an essential nutrient for healthy grapevine growth, excess accumulation in the berry can negatively influence wine quality (Mpelasoka et al. 2003). In the report to the GWRDC (CSL02/03) Kumar et al. (2006) clearly established that salinity, sodicity and available potassium were elevated in soils treated with WWW. Potassium originates from the grape skins and sodium predominantly from cleaning products eg. sodium citrate and sodium hydroxide. In Australia, a sodium adsorption ratio (SAR) of 6 is considered the benchmark of sodicity. Winery wastewater was found to be highly sodic (SAR = 10‐35) throughout the 2006 vintage (Kumar et al. 2006). Therefore, these elevated salt levels in WWW are a key limiting factor in its sustainable use in irrigation. Examples of current strategies to remove salts include reverse osmosis, which is expensive and clearly not economically viable or evaporation ponds, which results in no reuse of water. Biological removal of organic nutrients and salt (in particular phosphorus) has been studied in depth for domestic biological wastewater treatment systems (Seviour et al. 2003). For example, organisms that accumulate inorganic phosphorus in excess of their physiological requirements have been identified (eg. Candidatus ‘Accumulibacter phosphatis’) and their growth encouraged by anaerobic/aerobic cycling of the biomass, increasing the overall quality of effluent and decreasing its harmful impacts on the environment. This demonstrates that improvements to these systems can be made through a sound understanding of their microbiology. A similar approach was undertaken to investigate the use of halophiles to reduce the salt concentration in WWW. Halophiles are organisms that thrive in high salt environments. In order for these organisms to survive their cytoplasm has to be isoosmotic with their surrounding environment. There are 3 main strategies employed by organisms to achieve this: 1. Biosynthesis or accumulation of “compatible solutes”; organic osmotic solutes 2. Accumulation of intracellular potassium and chloride 3. A combination of mechanism 1 and 2 Strategy 2 is largely restricted to the “extreme halophiles”, those that require >2.5 M salt, much higher than WWW. Therefore in this study we targeted organisms that employ strategy 3, which includes moderate halophiles. Halophiles have been used in other industrial wastewater treatment plants to improve nutrient removal and remove colours (Kargi 2002). To date, no research has been carried out on WWW to investigate the presence and use of such potentially beneficial bacteria. The identification of 6 such organisms could lead to the development of a novel treatment process for WWW and allow the sustainable reuse of a key water resource. The aims of this project were to: 1. Study intracellular salt content in microorganisms from WWW through the use of molecular and fluorescent identification/screening techniques. 2. Investigate microorganisms capable of accumulating high levels of intracellular salt from the activated sludge of WWW. 3. Isolate and identify these organisms. 4. Explore the impact of inoculating WWW with wastewater isolates and halophiles known to accumulate high levels of salts. Project Outputs and Performance Targets Table 1. Project outputs and performance targets Outputs Performance Targets 1. Protocol for applying Staining and visualization of organisms with high fluorescent dyes to control intracellular Na/K levels. organisms and WWW biomass 2. Identification of organisms with Separation of organisms containing high intracellular high intracellular salt levels salt levels by flow cytometry and identification by gene sequencing. 3. Effect of dosing WWW with Compare salt concentrations of winery effluent in the known species of halophiles presence and absence of halophiles 4. Dissemination of project At least one conference presentation or publication findings. detailing the potential use of microorganisms to reduce salt concentrations. 7 Methods and materials: 1. Winery wastewater treatment plants examined in this study Wastewater samples (50 mL) were collected from plants in the Barossa Valley and one plant in the McLaren Vale, and kept on ice for no longer than 24 h before analysis. Each sample was microscopically (Olympus BH fitted with an Olympus DP50 digital camera) examined after Gram staining (Lindrea et al. 1999). 2. Growth of purchased halophiles The following strains were purchased and selected to reflect a large genetic diversity of halophiles. • ACAM591 Shewanella frigidimarinaT • ACAM35 Halomonas elongataT • ACAM547 Pseudoalteromonas haloplanktisT • DSMZ 13855 Salinibacter ruberT • DSMZ 2228 Halanaerobium praevalensT • DSMZ 3754 Halobacter salinarumT • DSMZ 17747 Marinobacter vinifirmusT • DSMZ